Dynamics and function of DNA methylation in plants.

DNA methylation is a conserved epigenetic modification that is important for gene regulation and genome stability. Aberrant patterns of DNA methylation can lead to plant developmental abnormalities. A specific DNA methylation state is an outcome of dynamic regulation by de novo methylation, maintenance of methylation and active demethylation, which are catalysed by various enzymes that are targeted by distinct regulatory pathways. In this Review, we discuss DNA methylation in plants, including methylating and demethylating enzymes and regulatory factors, and the coordination of methylation and demethylation activities by a so-called methylstat mechanism; the functions of DNA methylation in regulating transposon silencing, gene expression and chromosome interactions; the roles of DNA methylation in plant development; and the involvement of DNA methylation in plant responses to biotic and abiotic stress conditions.

Abiotic stress responses in plants.

Plants cannot move, so they must endure abiotic stresses such as drought, salinity and extreme temperatures. These stressors greatly limit the distribution of plants, alter their growth and development, and reduce crop productivity. Recent progress in our understanding of the molecular mechanisms underlying the responses of plants to abiotic stresses emphasizes their multilevel nature; multiple processes are involved, including sensing, signalling, transcription, transcript processing, translation and post-translational protein modifications. This improved knowledge can be used to boost crop productivity and agricultural sustainability through genetic, chemical and microbial approaches.

Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner.

Plants establish mutualistic associations with beneficial microbes while deploying the immune system to defend against pathogenic ones. Little is known about the interplay between mutualism and immunity and the mediator molecules enabling such crosstalk. Here, we show that plants respond differentially to a volatile bacterial compound through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. We found that exposure of Arabidopsis thaliana to a known plant growth-promoting rhizobacterium can unexpectedly have either beneficial or deleterious effects to plants. The beneficial-to-deleterious transition is dependent on availability of phosphate to the plants and is mediated by diacetyl, a bacterial volatile compound. Under phosphate-sufficient conditions, diacetyl partially suppresses plant production of reactive oxygen species (ROS) and enhances symbiont colonization without compromising disease resistance. Under phosphate-deficient conditions, diacetyl enhances phytohormone-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Therefore, diacetyl affects the type of relation between plant hosts and certain rhizobacteria in a way that depends on the plant's phosphate-starvation response system and phytohormone-mediated immunity.

Evolution and adaptation of terrestrial plant-associated Plantibacter species into remote marine environments.

Microbes are thought to be distributed and circulated around the world, but the connection between marine and terrestrial microbiomes remains largely unknown. We use Plantibacter, a representative genus associated with plants, as our research model to investigate the global distribution and adaptation of plant-related bacteria in plant-free environments, particularly in the remote Southern Ocean and the deep Atlantic Ocean. The marine isolates and their plant-associated relatives shared over 98% whole-genome average nucleotide identity (ANI), indicating recent divergence and ongoing speciation from plant-related niches to marine environments. Comparative genomics revealed that the marine strains acquired new genes via horizontal gene transfer from non-Plantibacter species and refined existing genes through positive selection to improve adaptation to new habitats. Meanwhile, marine strains retained the ability to interact with plants, such as modifying root system architecture and promoting germination. Furthermore, Plantibacter species were found to be widely distributed in marine environments, revealing an unrecognized phenomenon that plant-associated microbiomes have colonized the ocean, which could serve as a reservoir for plant growth-promoting microbes. This study demonstrates the presence of an active reservoir of terrestrial plant growth-promoting bacteria in remote marine systems and advances our understanding of the microbial connections between plant-associated and plant-free environments at the genome level.

The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism.

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

Correlation analysis between the amniotic fluid contamination and clinical grading of neonatal hypoxic-ischemic encephalopathy and biomarkers of brain damage.

BACKGROUND: Amniotic fluid contamination (AFC) is a risk factor for neonatal hypoxic ischemic encephalopathy (HIE); however, the correlation between AFC level and the incidence and clinical grading of HIE, in addition to relevant biomarkers of brain damage, have not been assessed. METHODS: This single-center observational study included 75 neonates with moderate-to-severe HIE. The neonates with HIE were divided into four subgroups according to the AFC level: normal amniotic fluid with HIE group (NAF-HIE), I°AFC with HIE group (I°AFC-HIE), II°AFC with HIE group (II°AFC-HIE), and III°AFC with HIE group (III°AFC-HIE). The control groups consisted of 35 healthy neonates. The clinical grading of neonatal HIE was performed according to the criteria of Sarnat and Sarnat. Serum tau protein and S100B were detected by enzyme-linked immunosorbent assay kits. Correlations of serum tau protein and S100B were evaluated using the Pearson correlation analysis. RESULTS: (1) The incidence of neonatal HIE in the NAF-HIE group was 20 cases (26. 7%), I°AFC-HIE was 13 cases (17.3%), II°AFC-HIE was 10 cases (13.3%), and III°AFC-HIE was 32 cases (42. 7%). The incidence of moderate-to-severe HIE in the I°-III°AFC-HIE groups was 73.3% (55/75). (2) In 44 cases with severe HIE, 26 cases (59.1%) occurred in the III°AFC-HIE group, which had a significantly higher incidence of severe HIE than moderate HIE (p < 0.05). In NAF-HIE and I°AFC-HIE groups, the incidence of moderate HIE was 45.2% and 29.0%, respectively, which was higher than that of severe HIE (X2 = 9.2425, p < 0.05; X2 = 5.0472, p < 0.05, respectively). (3) Serum tau protein and S100B levels in the HIE groups were significantly higher than in the control group (all p < 0.05), and were significantly higher in the III°AFC-HIE group than in the NAF-HIE and I°AFC-HIE groups (all p < 0.05). (4) Serum tau protein and S100B levels in the severe HIE group were significantly higher in the moderate HIE group (all p < 0.05). (5) Serum tau protein and S100B levels were significantly positively correlated (r = 0.7703, p < 0.0001). CONCLUSION: Among children with severe HIE, the incidence of III°AFC was higher, and the levels of serum tau protein and S100B were increased. AFC level might be associated with HIE grading.

GhMYB52 Like: A Key Factor That Enhances Lint Yield by Negatively Regulating the Lignin Biosynthesis Pathway in Fibers of Upland Cotton (Gossypium hirsutum L.).

In the context of sustainable agriculture and biomaterial development, understanding and enhancing plant secondary cell wall formation are crucial for improving crop fiber quality and biomass conversion efficiency. This is especially critical for economically important crops like upland cotton (Gossypium hirsutum L.), for which fiber quality and its processing properties are essential. Through comprehensive genome-wide screening and analysis of expression patterns, we identified a particularly high expression of an R2R3 MYB transcription factor, GhMYB52 Like, in the development of the secondary cell wall in cotton fiber cells. Utilizing gene-editing technology to generate a loss-of-function mutant to clarify the role of GhMYB52 Like, we revealed that GhMYB52 Like does not directly contribute to cellulose synthesis in cotton fibers but instead represses a subset of lignin biosynthesis genes, establishing it as a lignin biosynthesis inhibitor. Concurrently, a substantial decrease in the lint index, a critical measure of cotton yield, was noted in parallel with an elevation in lignin levels. This study not only deepens our understanding of the molecular mechanisms underlying cotton fiber development but also offers new perspectives for the molecular improvement of other economically important crops and the enhancement of biomass energy utilization.

The Isolation, Structural Characterization and Anti-Inflammatory Potentials of Neutral Polysaccharides from the Roots of Isatis indigotica Fort.

Polysaccharides have been assessed as a potential natural active component in Chinese herbal medicine with anti-inflammatory properties. However, the complex and indefinite structures of polysaccharides limit their applications. This study explains the structures and anti-inflammatory potentials of three neutral polysaccharides, RIP-A1 (Mw 1.8 × 104 Da), RIP-B1 (Mw 7.4 × 104 Da) and RIP-B2 (Mw 9.3 × 104 Da), which were isolated from the roots of Isatis indigotica Fort. with sequenced ultrafiltration membrane columns, DEAE-52 and Sephadex G-100. The planar structures and microstructures of RIP-A1, RIP-B1 and RIP-B2 were further determined by HPGPC, GC-MS, methylation analysis, FT-IR, SEM and AFM, in which the structure of RIP-A1 was elucidated in detail using 1D/2D NMR. The Raw 264.7 cells were used for the anti-inflammatory activity in vitro. The results showed that RIP-A1, RIP-B1 and RIP-B2 are all neutral polysaccharides, with RIP-A1 having the smallest Mw and the simplest monosaccharide composition of the three. RIP-A1 is mainly composed of Ara and Gal, except for a small quantity of Rha. Its main structure is covered with glycosidic linkages of T-α-Araf, 1,2-α-Rhap, 1,5-α-Araf, T-ß-Galp, 1,2,4-α-Rhap, 1,3,5-α-Araf and 1,6-ß-Galp with 0.33:0.12:1.02:0.09:0.45:11.41:10.23. RIP-A1 significantly inhibited pro-inflammatory cytokines (NO, TNF-α, IL-6 and IL-1ß) and increased anti-inflammatory cytokines (IL-4) in LPS-stimulated RAW 264.7 cells. Moreover, RIP-A1 could significantly inhibit the mRNA expression of TNF-α, IL-6 and L-1ß. It could also activate IKK, p65 and IκBα (the components of the NF-κB signaling pathway). In conclusion, the above results show the structural characterization and anti-inflammatory potentials of RIP-A1 as an effective natural anti-inflammatory drug.

Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis.

Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis.

The methyl-CpG-binding protein MBD7 facilitates active DNA demethylation to limit DNA hyper-methylation and transcriptional gene silencing.

DNA methylation is a conserved epigenetic mark that plays important roles in plant and vertebrate development, genome stability, and gene regulation. Canonical Methyl-CpG-binding domain (MBD) proteins are important interpreters of DNA methylation that recognize methylated CG sites and recruit chromatin remodelers, histone deacetylases, and histone methyltransferases to repress transcription. Here, we show that Arabidopsis MBD7 and Increased DNA Methylation 3 (IDM3) are anti-silencing factors that prevent gene repression and DNA hypermethylation. MBD7 preferentially binds to highly methylated, CG-dense regions and physically associates with other anti-silencing factors, including the histone acetyltransferase IDM1 and the alpha-crystallin domain proteins IDM2 and IDM3. IDM1 and IDM2 were previously shown to facilitate active DNA demethylation by the 5-methylcytosine DNA glycosylase/lyase ROS1. Thus, MBD7 tethers the IDM proteins to methylated DNA, which enables the function of DNA demethylases that in turn limit DNA methylation and prevent transcriptional gene silencing.

Three Novel Heterozygous Mutations of NPHS1 Gene Causing Infants with Congenital Nephrotic Syndrome: Two Chinese (Han) Cases.

BACKGROUND: Congenital nephrotic syndrome (CNS) of the Finnish type (CNF) is an autosomal recessively disorder. NPHS1 gene mutation is the main gene responsible for CNF. This study aimed to explore the clinical manifestations and the characteristics of genetic variation in Chinese patients with CNS. METHODS: A 15-minute-old boy and a 34-day-old girl with CNS were included. NPHS1 gene was detected by next-generation high-throughput sequencing. RESULTS: Patient 1 carried two novel heterozygous mutations of NPHS1 gene, one was c.204delG, p. (Leu69fs) in exon 2 of NPHS1 gene, a heterozygote frameshift mutation; the other was c.3558delT, p. (Gly1187fs) in exon 28, a heterozygote frameshift mutation. Patient 2 carried three heterozygous mutations of NPHS1, among them, c.1561-G>A. p.Asp521Asn in exon 12 is a heterozygous missense mutation. It was identified as possible de novo pathogenicity gene. CONCLUSIONS: Three novel heterozygous mutations of NPHS1 gene were responsible for the patients with CNS and can enlarge the spectrum of NPHS1 gene mutation.

Distribution and accumulation of cadmium in soil under wheat-cultivation system and human health risk assessment in coal mining area of China.

The soil contamination caused by the discharge of cadmium (Cd) from coal mining activities has aroused continuous attention due to the detrimental effects on the human health. This study aimed to investigate the characteristics on distribution of Cd in soils and its accumulation in wheat grains under wheat-cultivation system, and further assess the human health risks to adults and children. 58 soils and wheat samples in pairs from Linhuan coal mining area, Anhui Province were collected and analyzed. Results showed that the concentrations of Cd in 17.24% of soil samples exceeded the limit value established by the Ministry of Ecology and Environment. The ordinary kriging interpolation displayed that the spatial variability of Cd concentrations in soils was mainly influenced by coal mining activities. The transfer capacity of Cd from soils to wheat roots was greater than that from the wheat roots to grains. Multiple linear regression model clarified that soil pH and exchangeable Cd fraction in soils were the critical factors affecting the Cd accumulation in wheat grains. The carcinogenic risk of Cd levels in our studied wheat grains was a concern but still within the acceptable range, while their non-carcinogenic hazard was negligible for adults and children. The calculation results were in accord with the uncertainty analysis conclusion based on Monte Carlo simulation. The study was expected to promote the source management and control strategy of reducing tailing discharge, and providing scientific references for current soil remediation and land degradation prevention.

Genome-wide association study of ear tip barrenness in waxy maize.

Ear tip-barrenness (ETB), which results from aborted kernels or infertile florets at the ear tip, is an undesirable factor affecting the yield and quality of waxy maize. To uncover the genetic basis of ETB, a genome-wide association study (GWAS) was conducted using the genotype with 27,354 SNPs and phenotype with three environments. Five SNPs that distributed on chromosomes 1, 3 and 6, were identified to be significantly associated with ETB based on the threshold of false discovery rate (FDR) at 0.05. Among these significant loci, three SNPs were clustered together and colocalized with genomic regions previously reported. The average length of ETB decreased almost linearly from the inbred lines containing no favorable alleles across the three loci (1.75 cm) to those with one (1.18 cm), two (0.94 cm) and three (0.65 cm) favorable alleles. Moreover, three important genes, Zm00001d030028, Zm00001d041510 and Zm00001d038676 were predicted for three significant QTLs, respectively. These results promote the understanding genetic basis for ETB and will be useful for breeding waxy maize varieties with high-quality and high-yield.

Chromium in Chinese coals: geochemistry and environmental impacts associated with coal-fired power plants.

Chromium (Cr), one of the prime hazardous trace elements in coals, may engender adverse effects on eco-environment and threaten human health during utilization of coal. Based on the samples obtained in our laboratory and published literature, the abundance and modes of occurrence of Cr in Chinese coals, and the environmental impacts associated with coal-fired power plants (CFPPs) were elucidated in this study. With a total of 1397 sets of data, the mean concentration of Cr in Chinese coals was calculated as 21.33 µg/g by the "reserve-concentration" weighted calculation method. Spatially, the average Cr contents increased gradually from North China to South China. Temporally, coals from T3, E-N and P2 were relatively enriched in Cr compared to the other geological time. The Cr concentration in coal varied with different coal ranks. The geological factors accounted for Cr enrichment in coals could be divided into the primary, secondary and epigenetic processes. Higher percentages of organically Cr occurred in low-rank coals, while inorganically associated Cr was mainly found in clay minerals. After coal combustion, most of Cr was enriched in solid wastes (e.g., fly ash and bottom ash). The leaching of Cr from solid wastes in the rainy season (especially acid rain) needs to be a concern for CFPPs. It was estimated that the atmospheric emission of Cr from CFPPs increased annually from 2015 to 2019 and reached approximately 159 tons in 2019.

Aptamer-LYTACs for Targeted Degradation of Extracellular and Membrane Proteins.

Recently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N-acetylgalactosamine (tri-GalNAc) is a complex and time-consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer-based LYTAC (Apt-LYTAC) to realize liver-cell-specific degradation of extracellular and membrane proteins by conjugating aptamers to tri-GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt-LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt-LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

Phylogenetic analysis and character evolution of tribe Arethuseae (Orchidaceae) reveal a new genus Mengzia.

Delimitation of the tribe Arethuseae has varied considerably since it was first defined. The relationships within Arethuseae, particularly within the subtribe Arethusinae, remain poorly elucidated. In this study, we reconstructed the phylogeny of Arethuseae, using six plastid markers (matK, ycf1, rbcL rpoc1, rpl32-trnL and trnL-F) from 83 taxa. The ancestral state reconstruction of 11 selected morphological characters was also conducted to identify synapomorphies and assess potential evolutionary transitions. Morphological character comparision between the distinct species Bletilla foliosa and other species are conducted. Our results unequivocally supported the monophyly of Arethuseae, which included highly supported clades and a clear synapomorphy of non-trichome-like lamellae. Furthermore, B. foliosa formed a separate clade in the subtribe Arethusinae, instead of clustering with the other Bletilla species in the subtribe Coelogyninae. The morphological characters comparision further showed that the B. foliosa clade could be distinguished from other genera in Arethuseae by multiple characters, including presence of lateral inflorescence, three lamellae with trichome-like apex and four pollinia. In light of these molecular and morphological evidences, we propose Mengzia as a new genus to accommodate B. foliosa and accordingly provide descriptions of this new genus and combination.

Regulation of active DNA demethylation by an α-crystallin domain protein in Arabidopsis.

DNA methylation patterns are dynamically controlled by DNA methylation and active DNA demethylation, but the mechanisms of regulation of active DNA demethylation are not well understood. Through forward genetic screens for Arabidopsis mutants showing DNA hypermethylation at specific loci and increased silencing of reporter genes, we identified IDM2 (increased DNA methylation 2) as a regulator of DNA demethylation and gene silencing. IDM2 dysfunction causes DNA hypermethylation and silencing of reporter genes and some endogenous genes. These effects of idm2 mutations are similar to those of mutations in IDM1, a regulator of active DNA demethylation. IDM2 encodes an α-crystallin domain protein in the nucleus. IDM2 and IDM1 interact physically and partially colocalize at discrete subnuclear foci. IDM2 is required for the full activity of H3K18 acetylation but not H3K23 acetylation of IDM1 in planta. Our results suggest that IDM2 functions in active DNA demethylation and in antisilencing by regulating IDM1.

An Rrp6-like protein positively regulates noncoding RNA levels and DNA methylation in Arabidopsis.

Rrp6-mediated nuclear RNA surveillance tunes eukaryotic transcriptomes through noncoding RNA degradation and mRNA quality control, including exosomal RNA decay and transcript retention triggered by defective RNA processing. It is unclear whether Rrp6 can positively regulate noncoding RNAs and whether RNA retention occurs in normal cells. Here we report that AtRRP6L1, an Arabidopsis Rrp6-like protein, controls RNA-directed DNA methylation through positive regulation of noncoding RNAs. Discovered in a forward genetic screen, AtRRP6L1 mutations decrease DNA methylation independently of exosomal RNA degradation. Accumulation of Pol V-transcribed scaffold RNAs requires AtRRP6L1 that binds to RNAs in vitro and in vivo. AtRRP6L1 helps retain Pol V-transcribed RNAs in chromatin to enable their scaffold function. In addition, AtRRP6L1 is required for genome-wide Pol IV-dependent siRNA production that may involve retention of Pol IV transcripts. Our results suggest that AtRRP6L1 functions in epigenetic regulation by helping with the retention of noncoding RNAs in normal cells.

Histone acetylation recruits the SWR1 complex to regulate active DNA demethylation in Arabidopsis.

Active DNA demethylation is critical for controlling the DNA methylomes in plants and mammals. However, little is known about how DNA demethylases are recruited to target loci, and the involvement of chromatin marks in this process. Here, we identify 2 components of the SWR1 chromatin-remodeling complex, PIE1 and ARP6, as required for ROS1-mediated DNA demethylation, and discover 2 SWR1-associated bromodomain-containing proteins, AtMBD9 and nuclear protein X1 (NPX1). AtMBD9 and NPX1 recognize histone acetylation marks established by increased DNA methylation 1 (IDM1), a known regulator of DNA demethylation, redundantly facilitating H2A.Z deposition at IDM1 target loci. We show that at some genomic regions, H2A.Z and DNA methylation marks coexist, and H2A.Z physically interacts with ROS1 to regulate DNA demethylation and antisilencing. Our results unveil a mechanism through which DNA demethylases can be recruited to specific target loci exhibiting particular histone marks, providing a conceptual framework to understand how chromatin marks regulate DNA demethylation.